The present invention relates to methods for forming a peptide on a support matrix.
The Merrifield method of solid phase peptide synthesis is an extremely useful synthetic tool. In the Merrifield method, a peptide attached to a support matrix is lengthened by coupling with an amino acid. The N-terminus of the amino acid is protected by a blocking group such as a BOC group (t-butoxycarbonyl). The lengthened peptide is then decoupled by removing the blocking group, and then the coupling reaction is repeated. A problem with solid phase peptide synthesis is the determination of when the coupling and deprotection are complete. The completeness of these reactions is essential to peptide synthesis, since an incomplete coupling or deprotection reaction can lead to deletion of an amino acid in the desired sequence. These deletions can vary from trace to significant amounts.
Numerous methods for monitoring the completeness of the deprotection and coupling reactions have been reported. For example, the Kaiser (ninhydrin) test is convenient, rapid (requires about 5 minutes to run) and well documented. For these reasons, the Kaiser test is the most widely used of the qualitative monitoring methods. In the Kaiser test, a reagent is reacted with the unblocked supported peptide to produce a purple product, the intensity of the purple color qualitatively indicatng the amount of decoupling.
The Kaiser test has disadvantages. For example, it has been shown to give false positive results for complete coupling. In addition, the Kaiser test lacks sensitivity with respect to the degree of deprotection of BOC-amino acids because the intensity of Ruhemann's purple is noted from about 50 to about 100 percent free amino groups. Another drawback to the Kaiser test is its lack of good color resolution for the deprotection of secondary amino acids. More particularly, the deprotection of proline, hydroxyproline, and sarcosine gives a brown color instead of purple.
Another monitoring method uses chloromil. The chloromil method, like the Kaiser method, is inherently inaccurate since it relies on color differentiation. The reason for this is that in dilute solutions, faint amounts of color are difficult to detect with the naked eye.
Two common quantitative tests, the picric acid titration and the quantitative ninhydrin test, have advantages over the qualitative Kaiser and chloromil tests in that they give quantitative information about the degree of deprotection or coupling during peptide synthesis. However, both of these quantitative methods suffer as synthetic monitoring tools due to (1) the length of time (about 2 hours) required to complete the test because of the need to dry and accurately weigh the resin peptide samples; (2) the need for a highly skilled technician to obtain reproducible results; and (3) the lack of sensitivity in determining the completeness of the deprotection and coupling reactions. Since peptide chains can have 30 or more amino acids, a monitoring test that requires in excess of 2 hours per amino acid added to the support is not commercially feasible on a routine basis.
Accordingly, it would be very desirable to have a quantitative method for monitoring the completness of the coupling and deprotection reactions employed in solid phase peptide synthesis where the method (1) requires a relatively short period of time to perform; (2) does not require the use of a highly skilled technician to obtain reproducible results; and (3) is sensitive to the completeness of both the deprotection and coupling reactions.